Abstract KIR2DL4 (2DL4;CD158d) is a structurally and functionally unique member of the human killer cell Ig-like receptor (KIR) family. It can interact with HLA-G, which is normally expressed on trophoblast cells. In contrast to other KIR, engagement of 2DL4 can stimulate potent cytokine responses, but only weak cytotoxicity by freshly isolated NK cells. 2DL4 is only expressed on CD56high NK cells in peripheral blood, and is the only KIR that can be up-regulated upon culture in IL-2. The mechanisms regulating expression and function of 2DL4 remain poorly understood. Interestingly, common alleles of the 2DL4 gene encode proteins that cannot reach the surface of NK cells, and as many as 25% of the human population are homozygous for non-expressed alleles. This suggests that these individuals may lack physiologically important innate immune responses that may make them more or less susceptible to certain disease states. We have established that the unique activating properties of 2DL4 are mediated through two distinct signaling modules that are dependent or independent of physical association with the Fc[unreadable]RI-[unreadable] signaling adaptor. Furthermore, we have recently discovered that 2DL4 can associate with an important kinase and an E3 ubiquitin ligase that can mediate and regulate receptor function, respectively. Finally, we have evidence for a novel ligand expressed on transformed epithelial and fibroblast cell lines that has potential to engage 2DL4 at sites of cancer or inflammation. We propose to define the molecular basis by which KIR2DL4 can activate distinct functional response programs in NK cells, which may occur when they encounter trophoblasts, tumor cells, or inflammatory sites by pursuing the following specific aims: Aim 1. How does an E3 ubiquitin ligase regulate KIR2DL4 expression and function? Aim 2. What is the mechanism of Fc[unreadable]RI-[unreadable]-independent signaling by KIR2DL4? Aim 3. Characterization of novel KIR2DL4 ligand(s) on transformed epithelial and fibroblast cells